Project Summary Voltage-gated sodium channels (Navs) are targets of a variety of natural neurotoxins and insecticides, including pyrethroids and sodium-channel-blocker insecticides (SCBIs). Pyrethroids are currently one of the most important and effective arsenals against mosquitoes and other arthropod vectors that transmit human diseases. SCBIs belong to a newer class of selective insecticides. A major challenge for effective vector control is global emergence of insecticide resistance. In particular, mutations in Nav genes confer a major form of resistance known as knockdown resistance (kdr). In the absence of 3-D structures of eukaryotic Navs in complex with pyrethroids or SCBIs, major uncertainties remain in understanding their interactions (including the nature of respective receptor sites) and kdr mechanisms. In this grant proposal, we take advantage of recent breakthroughs in the co-PI?s laboratory in the determination of the cryo-EM structures of several eukaryotic Navs. The availability of these Nav structures now promises an unprecedented new level of understanding of Nav interactions with insecticides and other neurotoxins. We propose to determine cryo-EM structures of Navs in complex with pyrethroids, SCBIs and three other neurotoxins and use these structures as a guide to conduct a comprehensive series of mutational, electrophysiological and computational modeling analyses of respective Nav-ligand interactions. Two specific aims of this renewal proposal are: 1) determine cryo-EM structures of eukaryotic Nav complexes with various pyrethroids to gain insights into the atomic mechanisms of pyrethroids and kdr, and 2) Investigate the mechanisms of Nav interactions with SCBIs (DCJW and metaflumizone), steroidal agonists (BTX and veratridine) and the alkylamide insecticide BTG 502. Results from this research will push our understanding of the molecular actions of pyrethroids, SCBIs and other neurotoxins to a new level, thereby facilitating the development of precision monitoring and management of insecticide resistance worldwide and discovery of new and safe Nav-targeting chemicals to combat global threats from arthropod disease-transmitting vectors.